KR20130115327A - Method for evaluation of regenerated cartilage - Google Patents

Abstract

The method for evaluating regenerated cartilage according to the embodiment includes leaving the cell group including the ear cartilage cells in the presence of a medium, and then collecting at least a portion of the liquid component from the medium, and including the collected liquid component in the collected liquid component. Measuring GFAP content, and determining whether regenerated cartilage obtained or obtainable from the cell population is suitable for transplantation based on the GFAP content.

Description

Evaluation method of regenerative cartilage {METHOD FOR EVALUATION OF REGENERATED CARTILAGE}

The present invention relates to a method for assessing regenerated cartilage.

Tissue repair and reconstruction by autologous transplantation have been performed so far about congenital morphogenesis abnormalities in the maxillofacial surface and maxillofacial defects after malignant tumor resection. However, autologous tissue transplantation remains a problem such as a limitation of the size of tissue that can be harvested, or an invasion to a site to be transplanted. Among them, cartilage regenerative medicine has been comparatively advanced in clinical applications. Currently, autologous chondrocyte implantation (ACI) using autologous chondrocytes has been widely used worldwide. The ACI Law was first reported in 1995 by a Swedish research group. The ACI method is a method of administering autologous chondrocytes to a defect as a cell suspension for local defects of articular cartilage caused by sports trauma and the like, and coating with a periosteal patch to prevent leakage.

Defects and abnormalities in the maxillofacial region have been reported by application of a suspension of autologous chondrocytes to cases of rejuvenation surgery or ophthalmology with silicone implants.

For example, gene expression and surface markers have been used to characterize cultured chondrocytes. However, they are lacking in quantitation, and it is a phenomenon that it is necessary to sacrifice part of the cell population to be finally obtained.

Related arts of such techniques are described in the literature, for example.

Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. 1994. Treatment of deep cartilage defect in the knee with autologous chondrocytes transplantation. N Engl J Med 331 (14): 889-895. Yanaga H, Yanaga K, Imai K, Koga M, Soejima C, Ohmori K. 2006. Clinical application of cultured autologous human auricular chondrocytes with autologous serum for craniofacial or nasal augmentation and repair. Plast Reconstr Surg 117 (6): 2019-2030.

It is an object of the present invention to provide a method for quantitatively evaluating regenerated cartilage without sacrificing a part of regenerated cartilage or a portion of chondrocytes serving as a material thereof.

According to one aspect of the present invention, the cell group including the canine chondrocytes is left in the presence of a medium, and then at least a part of the liquid component is collected from the medium, and the GFAP content contained in the collected liquid component. There is provided a method for evaluating regenerated cartilage comprising determining the regenerated cartilage obtained or obtainable from the cell population based on the GFAP content and determining whether the regenerated cartilage is suitable for transplantation.

1 is a diagram showing the expression level of GFAP gene in various cells.
Fig. 2 shows the expression levels of GFAP genes in human ear chondrocytes with different origins.
3 is a diagram showing the results of evaluation of the correlation between GAG and GFAP produced by human ear chondrocytes using cultured human ear chondrocytes.
4 is a diagram showing the results of evaluation of the correlation between COL2 and GFAP produced by human ear chondrocytes using cultured human ear chondrocytes.
Fig. 5 shows the results of evaluation of the correlation between GAG and GFAP produced by human ear chondrocytes using a culture supernatant obtained by culturing human ear chondrocytes.
Fig. 6 shows the results of evaluating the correlation between COL2 and GFAP produced by cultured human ear chondrocytes using the culture supernatant obtained by culturing human ear chondrocytes.
Fig. 7 shows the results of measurement of the amount of GFAP in the culture supernatant obtained by culturing each of chondrocytes and fibroblasts.
FIG. 8 is a view showing an image of regenerated tissue after transplantation of cultured chondrocyte # 1 shown in FIG. 7.
FIG. 9 is a view showing an image of regenerated tissue after transplantation of cultured chondrocyte # 2 shown in FIG. 7.
FIG. 10 is a view showing an image of regenerated tissue after transplantation of cultured chondrocyte # 3 shown in FIG. 7.
FIG. 11 is a view showing an image of regenerated tissue after transplantation of cultured fibroblast # 1 shown in FIG. 7.
FIG. 12 is a view showing an image of regenerated tissue after transplantation of cultured fibroblast # 2 shown in FIG. 7.
FIG. 13 is a view showing an image of regenerated tissue after transplantation of cultured fibroblast # 3 shown in FIG. 7.
14 is a view schematically showing a manufacturing process of the ear cartilage regeneration product according to one embodiment.

1. Overview

Conventionally, as a regenerative medicine for rebuilding cartilage, a method of injecting chondrocytes has been performed.

However, since chondrocytes themselves do not have sufficient size and sufficient strength, there is still a high possibility that infusion of chondrocytes alone will not fully serve the purpose of tissue reconstruction. In this situation, the present inventors have developed a means for providing regenerated cartilage such as an "implant" having sufficient size or strength. In the development, first of all, it was noted that when chondrocytes are transplanted into the body, it is necessary to ensure a certain quality. Specifically, this quality means that the regenerated cartilage has cartilage characteristics. The term "cartilage properties" is used interchangeably with words of "characteristics as cartilage", "characteristics of cartilage", "characteristics as cartilage", and "characteristics of cartilage". "Cartilage characteristics" means specific properties as cartilage suitable for transplantation. Specifically, it refers to a property that can be safely transplanted, cartilage is formed after transplantation, and can be engrafted as cartilage in the body. According to an example, "cartilage characteristic" means the property which shows the cartilage regeneration vigorously in the body after transplantation. In addition, "cartilage characteristics" may be interpreted as transplantation feasibility. In order for regenerated cartilage to have cartilage properties, it is necessary that there is no incorporation of foreign substances, such as other cells, and that it does not overgrow.

Chondrocytes are an important component of the secretion of cartilage substrate, which determines the properties and functions of regenerated cartilage. For example, when the chondrocytes overproliferate, the cartilage properties are weakened by dedifferentiation, and further, the cartilage properties are lost. In addition, when fibroblasts are mixed when chondrocytes are isolated and the fibroblasts overproliferate in culture, there is a risk in the manufacturing process that constructs the desired chondrocytes. When cultured tissue in which chondrocytes are constructed by fibroblasts is transplanted, fibroblasts are transplanted instead of chondrocytes. Such transplanted tissue cannot engraft as cartilage. Therefore, the regenerated cartilage product produced can contain cartilage cells in a sufficient amount without containing other cells in an amount sufficient to build up the chondrocytes, and can maintain cartilage properties after transplantation without overproliferation. It is indispensable to determine beforehand.

The inventors have found specific markers which can be obtained by using a population of cells including chondrocytes, in particular ear chondrocytes, or which are indicative of whether the regenerated cartilage obtained is suitable for transplantation. The present invention is based on this finding. According to this marker, it is possible to quantitatively evaluate the cartilage properties of the regenerated cartilage that can be obtained or obtained using the cell population without sacrificing the final target product to be obtained.

2. Markers Specific to the Auricular Chondrocytes

A marker specific for the above described chondrocytes, in particular the chondrocytes, is Glial fibrillary acidic protein (GFAP).

GFAP is a protein generally localized specifically in the nervous system, such as astrocytes or Schwann cells.

The inventors have found that GFAP expresses specifically and in high amounts in cultured chondrocytes, which are useful cell sources of cartilage regeneration medicine, and the expression level of GFAP gene and GFAP protein gradually depending on subculture. The quantity decreased.

By quantifying the amount of this GFAP protein, it is possible to detect the incorporation of other cells such as fibroblasts, attenuation of cartilage properties due to overproliferation of the ear chondrocytes, and the like. This provides a method for evaluating cartilage properties of regenerated cartilage products.

According to one aspect of the present invention, there is provided a method for leaving a cell population including an open chondrocyte in the presence of a medium, thereafter separating at least a portion of the liquid component from the medium, and GFAP contained in the separated liquid component. There is provided a method for evaluating regenerated cartilage comprising measuring the content and determining whether regenerated cartilage obtained from the cell group is suitable for transplantation based on the GFAP content.

In this method, it is possible to quantitatively obtain information on the accumulation amount of cartilage substrate by measuring the content of GFAP contained in a liquid component such as the culture supernatant.

As used herein, the term "cartilage substrate" refers to a substance that is the basis of cartilage structure. Cartilage substrates are, for example, glycosaminoglycans (GAG), type II collagen (COL2).

As shown in the examples to be described later, in a study based on the present invention, it was found as a new finding that the GFAP content in the culture supernatant correlated with the accumulation amount of GAG and COL2. Based on these findings, it has become possible to evaluate cartilage properties by measuring the content of GFAP in a liquid component such as culture supernatant.

For example, in human articular chondrocytes used for the production of regenerated cartilage, CD10, CD26, CD44, CD49c, CD81, CD166, and the like, as markers for enhanced expression, include CD94a, CD106 and the like. These are cell adhesion molecules and are used as cell surface markers. Cell surface markers are interpreted by flow cytometry and it is difficult to quantitatively compare different systems interpreted separately. In addition, there is also a possibility of fluctuation depending on the culture environment such as the adhesion state of the cells, and it is difficult to provide a constant standard.

In addition to the cell adhesion molecules, serine protease inhibitors A1 and A3, fibroblast growth factor-18 (FGF-18) and melanoma inhibitory activator (MIA) have been identified. In particular, FGF-18 may be used as an index indicating the substrate production ability of chondrocytes because it is a factor that increases the gene expression amount by repeated planar culture. However, when these factors are used as an index, since the evaluation is performed at the gene level, the problem remains in quantitative. Also, they are all secreted proteins. It is expected that the bioactive substance secreted from cultured chondrocytes during proliferation is extremely small, and the amount of protein contained therein may be measured using the culture supernatant as a sample, but it is difficult to detect. For this reason, GFAP was adopted as an index.

GFAP has a smaller expression amount in articular chondrocytes than small ear chondrocytes, and a small change in expression amount due to passage. Therefore, as a source of regenerated cartilage, an ear chondrocyte is used.

For the above reason, it is clear that the evaluation method according to one embodiment of using an ear chondrocyte as a source of regenerated cartilage and using GFAP as an index of cartilage properties of regenerated cartilage is advantageous.

3. Chondrocytes

The chondrocytes used here are the ear chondrocytes.

The source of chondrocytes may be any mammal. Mammals are, for example, mice, rats, rabbits, dogs, cats, horses, cows, monkeys and humans, preferably humans.

The mammal that is a source of chondrocytes is selected depending on the mammal to be transplanted with the regenerated cartilage composed of chondrocytes. Description of regenerated cartilage composed of chondrocytes will be described later.

When a human is a target of transplantation, it is an alien chondrocyte derived from a human (hereinafter "human ear chondrocyte"), More preferably, it is an autologous ear chondrocyte derived from a human. Herein, "autologous chondrocytes" refers to cells collected prior to the production of transplanted and regenerated cartilage from the target of the subject to be transplanted with regenerated cartilage composed of chondrocytes.

Chondrocytes are isolated, for example, as follows. First, cartilage pieces are surgically collected from a donor mammal. Next, the collected cartilage pieces are fragmented under aseptic conditions and digested with the desired digestive enzymes such as collagenase and / or gelatinase, trypsin, pepsin and agrecanase, as necessary. Thereby, a cell population including at least a part of the cell population including each of the cartilage cells which are individually released and / or the microstructure fragments formed by the plural chondrocytes which are combined is obtained.

4. Cultivation of Chondrocytes

Subsequently, the cell group including the canine chondrocytes is left in the presence of the medium.

Here, the medium may be cell division or may not be cell division. For example, the medium is a buffer containing a component necessary for the survival of the cell population.

"Neglect" means to maintain a cell under specific conditions. In this maintenance, cell division typically occurs. That is, the culture may be left to stand. In this maintenance, cell division does not have to occur. For example, if the cartilage viable cells necessary for the production of regenerated cartilage have already been obtained, cell division may not occur in this maintenance. In the case of cell division in maintenance under specific conditions, that is, in the case of culturing, the method of culturing may be any method known to those skilled in the art so long as the number of viable cartilage cells necessary for producing regenerated cartilage can be obtained. have. The method of culturing may be, for example, passage in order to obtain the required live cell number. Passage culture is performed by culture | cultivating isolated chondrocytes by planar culture for 3 weeks, passing once, culture | cultivating for 3 weeks, and multiplying by about 1000 times according to an example. In addition, additional passage culture may be performed. There is no restriction | limiting in particular in the form of culture, According to the objective, it can select suitably. The form of the culture is, for example, suspension culture in a bottle, planar and / or suspension culture (monolayer culture) in a culture dish, pellet culture, three-dimensional culture by hydrogel and the like.

The medium used is a medium suitable for the culture of chondrocytes known per se. The medium is preferably DMEM, MEM, DMEM / F12 and serum-containing DMEM / F12. The amount of the medium is 20 to 20,000 mL with respect to 1 mL of the volume of the cell group including the chondrocytes.

For example, leaving is performed at a humidity of 80 to 100% and a temperature of 36 ° C to 38 ° C in the presence of 3 to 7% of CO ₂ . The time of standing is time required for culturing, for example, when culturing is performed. In addition, although it is not necessary to carry out stirring a medium during standing, stirring is preferable.

The cell group including the chondrocytes after the above-mentioned standing may form a tissue at least partially by binding to each other, or may be cartilage cells individually released. The cell group including chondrocytes may be a primary culture and / or a passage culture.

The survival rate of chondrocytes included in the cell group, that is, the ratio of cartilage living cells in the chondrocytes included in the cell group, is preferably 80% or more. If the survival rate of chondrocytes is less than 80%, there is a possibility that healthy cartilage regeneration is not expected. The method of measuring the survival rate is any method known per se. The method of measuring the survival rate is, for example, a PE method using a Nucleocounter.

The cell population including chondrocytes contains, for example, more than 240 million chondrocytes. This cell group contains the number of live cartilage cells necessary for the production of regenerated cartilage, and can be used to produce regenerated cartilage.

5. Measurement of GFAP amount

Subsequently, at least a part of the liquid component is collected from the medium described above. Here, the liquid component can be obtained by removing at least the cells from the medium. The culture medium may be a culture medium, or may be a buffer solution in which the medium is temporarily replaced with a buffer having a physiological composition known per se, and further incubated for a desired time. The liquid component is, for example, culture supernatant.

As a method of extracting a liquid component from a medium, any method of obtaining a medium so that a cell group is not contained from a medium is used. For example, a centrifugal separation method is used as a method of collecting the liquid component from the medium.

The time for collecting the liquid component from the medium is, after isolation of the cell group including chondrocytes, before cultivation, during cultivation, after cultivation, at the end of cultivation, during primary culture, before subculture, after subculture, during subculture, and during regeneration cartilage. At least one time period selected from before formation, after regeneration cartilage formation, before transplantation of regeneration cartilage and after transplantation of regeneration cartilage. For example, immediately after isolating a cell group including chondrocytes, the cell group is suspended in a buffer, and the liquid component in the suspension obtained is collected. When a liquid component is taken immediately after isolation and used to measure the amount of GFAP, the purity of chondrocytes in the isolated cell group can be examined. In addition to immediately after isolation of the cell population, for example, during the culture and / or after the formation of regenerated cartilage, a liquid component may be taken immediately before transplantation and provided for the following measurements. The quality of regenerated cartilage can be controlled when liquid components are taken immediately before implantation and given to the measurement.

Next, the GFAP content contained in the liquid component separated in this way is measured. The method for measuring the GFAP content is any method known per se that can measure the amount of GFAP. For example, the method of measuring GFAP content is ELISA method, PR-PCR method, and western blotting method.

Subsequently, based on the GFAP content measured as described above, it is determined whether the regenerated cartilage that can be obtained or obtained from the cell population is suitable for transplantation. For example, when the GFAP content contained in the liquid component is 0.05 ng or more per mL of the liquid component, it is determined that the regenerated cartilage that can be obtained or obtained from the cell population is suitable for transplantation. Preferably, when the GFAP content contained in the liquid component is 0.5 ng or more per mL of liquid component, it is judged that the regenerated cartilage obtainable or obtained from the cell population is suitable for transplantation. More preferably, when the GFAP content included in the liquid component is 5 ng or more per mL of the liquid component, it is judged that the regenerated cartilage obtained or obtained from the cell population is suitable for transplantation.

If the GFAP content contained in the liquid component is 0.05 ng or more per mL of the liquid component, the regenerated cartilage that can be obtained or obtained from the cell population is engrafted as cartilage after transplantation, and preferably exhibits strong cartilage regeneration. On the other hand, when the GFAP content is less than 0.05 ng per mL of liquid component, the regenerated cartilage that can be obtained from the cell group is likely to not engraft as cartilage or exhibit desired cartilage regeneration after transplantation.

The cell group determined to be suitable for transplantation is then used for formation of regenerated cartilage described below. On the other hand, the cell population judged not suitable for transplantation is not used for formation of regeneration cartilage.

In addition, when the above-mentioned evaluation is performed on the regenerated cartilage, and it is determined that the regenerated cartilage is not suitable for transplantation, the regenerated cartilage is not transplanted.

6. Formation of Regenerative Cartilage

Regenerated cartilage is formed using a cell population including chondrocytes left in the presence of a medium. The cell group used here is a cell group judged that the regeneration cartilage which can be obtained from a cell group is suitable for transplantation based on GFAP content as mentioned above.

For example, regenerated cartilage is formed by three-dimensional culture of a cell group including chondrocytes. Three-dimensional culture is carried out using a scaffold material. Specifically, three-dimensional culture is carried out by culturing chondrocytes on or inside the scaffold material. The material, property, shape, structure, size, etc. of the scaffold material used are not limited, and can be appropriately selected according to the purpose. Scaffold materials are, for example, atelocollagen, fibrin, hyaluronic acid and biodegradable polymeric porous bodies. Biodegradable polymeric porous bodies are, for example, PLLA, PGA and PLGA. The scaffold material can be formed using any known method. Three-dimensional culture may also be performed using a gelling agent. For example, by mixing a gelling agent and a cell group including chondrocytes and contacting the scaffold material, it is possible to perform stable three-dimensional culture to obtain stable regenerated cartilage. Gelling agents are, for example, atelocollagen and alginate. Three-dimensional culture is carried out, for example, at 37 ° C. in a 5% CO ₂ incubator under 2 hours of conditions.

As described above, regenerated cartilage suitable for transplantation is produced.

It is preferable that the regenerated cartilage thus produced has a certain size and can be used as an implant. The regenerated cartilage may be in suspension form or in solid form, but is preferably in solid form.

The regenerated cartilage produced can be used, for example, as a medical implant for supplementing or reinforcing cartilage defects or cartilage damage sites. In particular, it can be used as a medical graft material for congenital morphogenesis abnormalities, such as nasal deformity of cleft lip and palate. The regenerated cartilage may also be used as a cosmetic material for implantation.

Cartilage regenerative medicine mainly includes four processes: (1) cartilage extraction from patients, (2) isolation and culture of chondrocytes, (3) recovery of cultured chondrocytes or formation of regenerated cartilage, and (4) transplantation into patients. It includes. Isolation and cultivation of the second chondrocyte is an essential part of the manufacture of regenerative medical products carried out in special facilities called cell processing centers. Therefore, it is also a process that requires the strictest quality control.

Conventionally, due to the incorporation of cells other than chondrocytes or over-proliferation of chondrocytes, the quality of the regenerated cartilage is not guaranteed and the patient cannot provide the prescribed medical care or the cartilage formation is reduced after transplantation. The incorporation of other cells could not be determined because the index indicating the purity of chondrocytes was not established. Other cells interfere with the proliferation of chondrocytes and the differentiation of cell populations into cartilage. In addition, over-proliferation of chondrocytes significantly progresses the dedifferentiation of cultured cell populations, resulting in attenuation of characteristics as cartilage. Hyperproliferation has arisen because accurate measurement of the chondrocyte count at the time of isolation is difficult. In other words, the number of chondrocytes at the time of isolation was excessively estimated, and the target cell numbers were obtained by subculture, resulting in over-proliferation of chondrocytes.

According to the method for evaluating the regenerated cartilage described above, the cartilage properties of the regenerated cartilage obtained or obtainable from the cultured chondrocytes can be evaluated, thereby solving the problems which have arisen in the past as described above. Specifically, the evaluation method can obtain a quantitative index indicating the purity of chondrocytes in the cell population by measuring the GFAP content. Can be excluded. And since it is judged whether the regeneration cartilage manufactured using the cell group containing chondrocytes is suitable for transplantation, the situation that cartilage formation falls in the body after transplantation can be avoided.

In addition, for evaluation of cartilage characteristics, a cell group containing chondrocytes is not used, but a liquid component such as culture supernatant that is separated from the medium and does not contain chondrocytes is used. Therefore, it is very advantageous in the production of regenerated cartilage in that it does not damage the cell which becomes a valuable raw material of regenerated cartilage products.

In addition, the quality of the regenerated cartilage produced using the above-described method of evaluating cartilage characteristics is guaranteed. Therefore, such regenerated cartilage can be safely used as a medical and cosmetic transplant material.

[Yes]

Hereinafter, examples of the present invention will be described.

≪ Example 1 >

 Example 1. Examination of the indicator for determining that the constituent cells of the regenerated cartilage product are the fibrocartilage cells

The gene which expresses highly in the earliest chondrocytes multiplying 1000 times after the culture used for manufacture of regenerated cartilage product was searched by the following method.

Forcible chondrocytes, fibroblasts, cutaneous keratinocytes, organ epithelial cells, articular chondrocytes, costal chondrocytes, and astrocytes are passaged once a week by the monolayer culture method, followed by long-term continuous passage. did. Total RNA was collected from each of the cultured cells at the time points of the second passage (PS) and the eighth passage (P8), and microarray method using GeneChip® Human Genome U133 plus Array manufactured by Affymetrix was used for each cell. We searched for genes that are highly expressed.

As a result, genes such as fibroblast growth factor (FGF) -18 and glia cell fibrous acidic protein (GFAP) were detected, but among them, the gene with particularly high expression was the GFAP gene.

In addition, the expression level of the GFAP gene was measured by the Real Time PT-PCR method. Three samples were measured for each cell, and the mean and standard deviation of the measured values were obtained.

The results obtained for each cell are shown in FIG. 1.

From Fig. 1, it was shown that the expression level of GFAP was significantly higher in the chondrocytes among the chondrocytes, fibroblasts, cutaneous keratinocytes, organ epithelial cells, articular chondrocytes, costal chondrocytes and astrosite.

From these results, it was suggested that by detecting GFAP, the risk of fibroblasts being built up by fibroblasts during culture and unintentionally administered fibroblasts can be eliminated.

Example 2. Examination of Aptitude as an Indicator of Cartilage Substrate Production of GFAP Gene

Six cases of canine chondrocytes of different origin were passaged about once a week in DMEM / F12 culture medium containing 5% serum and FGF-2 and insulin, and long-term continuous passage was performed to monolayer culture (P8). Total RNA was collected from cultured cells at 100-fold increase of fibroblasts (P2) and 100 million-fold cells (P8) after cultivation of abundant substrate, and the expression of GFAP gene was expressed by Real Time PT-PCR method. Measured by.

The results are shown in FIG. From FIG. 2, it was shown that the expression of P2 was high in all the cases, but despite being cultured under the same culture conditions, the gene expression in P2 was 7.2 × 10 ³ to 1.321 × 10 ⁶ by the number of copies, between cells. It turns out that the gap is very big.

Here, the evaluation at the protein level was examined in order to suppress the gap in measurement.

Example 3. Measurement of Expression of GFAP at Protein Level

The donor was passaged once a week in 5% serum and FMEM-2 and insulin-containing DMEM / F12 cultures to another 18 human ear chondrocytes, and the organs were cultured to the third passage. Cultured canine chondrocytes were collected by trypsin treatment, and mixed with 1% atelocollagen gel at a concentration of 1 × 10 ⁷ cells / mL. 20 µl of this cell-mixed gel was divided into tubes and BMP-2 / insulin / containing bone morphogenetic protein-2 (BMP-2), insulin, and thyroid hormone (T3 (Triiodothryonine)). High-density embedding culture was performed in T3-containing chondrocyte substrate production-inducing medium, and after 3 weeks, the amount of GAG and COL2 secreted by chondrocytes was measured by colorimetric method and ELISA method. The same P3 cultured chondrocytes were collected from 1 × 10 ⁴ cells, and the amount of GFAP in the cell lysate was measured by ELISA method.The same test was performed on 18 cases of donor chondrocytes with different donors to secrete the cultured GAG. The correlation between the amount, the amount of COL2 and the amount of GFAP in the cells was calculated by Excel statistics manufactured by SSRI, Inc. The results are shown in Figs.

3 and 4, the correlation coefficient between the amount of GFAP and GAG in the cell is 0.507, the correlation coefficient between COL2 is 0.590, and there is a moderate correlation between the two values. From this, it was suggested that the functional evaluation of chondrocytes becomes possible by measuring the GFAP of chondrocytes constituting regenerated cartilage at the protein level.

Example 4. Examination of Sample to Evaluate GFAP Content

The donor cultured the other 18 cases of the earliest chondrocytes up to the third passage (P3), the cultured ear chondrocytes were collected by trypsin treatment, and the 1% atelocollagen gel was collected at a concentration of 1 × 10 ⁷ cells / mL. Mixed. 20 microliters of this cell-mixed gel was divided | segmented into the test tube, and high density embedding culture was performed in the BMP-2 / insulin / T3 containing chondrocyte production | generation induction medium. After 3 weeks, the amount of GAG and COL2 secreted by chondrocytes were measured by colorimetric method and ELISA method. In addition, a portion of the culture supernatant of the same P3 cultured chondrocytes embedded in atelocollagen was collected, and the amount of GFAP contained in the culture supernatant was measured by ELISA method. The donor performed the same test with 18 different ear chondrocytes, and calculated the correlation between the amount of GAG secreted by the cultured cells, the amount of COL 2 and the amount of GFAP in the culture supernatant. The results are shown in FIGS. 5 and 6.

5 and 6, the correlation coefficient between the amount of GFAP and GAG in the culture supernatant was 0.767, the correlation coefficient between COL2 was 0.735, and there was a strong correlation between the two values. In addition, the higher the GFAP content in the culture supernatant, the more vigorous cartilage regeneration was observed.

Therefore, by extracting a part of the culture supernatant and measuring the amount of GFAP by ELISA, it was found that shipment can be determined without damaging cells or tissues.

Example 5. Determination of GFAP Protein Amount for Evaluation of Cartilage Characteristics

Culture of second passage (P2) Three cases of cultured chondrocytes and cultured fibroblasts were cultured for 1 week in DMEM / F12 culture medium containing 5% serum and FGF-2 and insulin in cartilage proliferation medium. 1 mL of culture supernatant was fractionated from this cultured cell, and cultured cell was collect | recovered by the trypsin process after that. The recovered cells were mixed with 1% atelocollagen gel at a concentration of 1 × 10 ⁸ cells / mL to the porous scaffold material, and incubated at 37 ° C. for 2 hours to gel. It was implanted subcutaneously in the back of nude mice.

Two months later, the implants were removed, divided in half, and one side was used for histological evaluation by toluidine blue staining. The other one was used for quantitative evaluation of protein of GAG amount and type II collagen amount. The amount of GFAP contained in the culture supernatant collected was measured by ELISA method. The results are shown in FIGS. 7 and 8-13.

From the results of the amount of GFAP in the culture supernatant of Fig. 7 and histological evaluation of Figs. 8 to 13, it was shown that in all examples of the fibrocartilage cells, GFAP was detected from the culture supernatant and cartilage formation was observed by nude mouse transplantation. In fibroblasts, GFAP was not detected in all cases, and cartilage formation was not seen.

7 to 13 show that in cultured human immature chondrocytes that have cartilage after transplantation and show strong cartilage regeneration, GFAP is present at least 0.05 ng / mL or more in the culture supernatant. From these, when GFAP is contained in the culture supernatant of 0.05 ng / mL or more, it is suggested that cultured human ear cartilage cells forming regenerated cartilage is contained, and the value of the amount of GFAP contained in the culture supernatant is determined by the quality of regenerated cartilage. It became clear that it could be used as a standard.

<Example 2>

According to the following procedure, regenerated cartilage was produced. 14 is a diagram schematically illustrating a manufacturing process of the regenerated cartilage.

A part of the patient ear cartilage (approximately 1 cm x 5 mm x 2 mm) was taken, and chondrocytes were isolated by collagenase treatment. Isolated chondrocytes are incubated for about 3 to 6 weeks in 5% autologous serum and FGF-2, insulin containing DMEM / F12 culture. The cells were cultured to recover cells that had grown to a cell number of about 1000 times. The harvested cultured chondrocytes were administered to a PLLA scaffold material to form regenerated cartilage (FIG. 14).

On the other hand, the protein concentration of GFAP contained in the culture supernatant was measured using ELISA as a culture solution test 39 days after culture (3 days before transplantation). At 39 days (3 days before transplantation) after culturing, it was determined that GFAP was contained in 0.05 ng / mL or more in the culture supernatant, so that transplantation was possible safely, cartilage was formed in the body after transplantation, and engraftment was possible as regenerated cartilage. .

Claims (7)

Leaving a cell group including the ear chondrocytes in the presence of a medium,
Thereafter, extracting at least a portion of the liquid component from the medium,
Measuring the content of GFAP contained in the collected liquid component, and
Determining whether regenerated cartilage obtained or obtainable from the cell population is suitable for transplantation based on the GFAP content
Evaluation method of regenerated cartilage comprising a. The method of claim 1,
Said judgment is an evaluation method of regenerated cartilage which judges that it is suitable for transplantation, when the said GFAP content is 0.05ng or more per 1 mL of said liquid components. The method of claim 1,
Said judgment is an evaluation method of regenerated cartilage which judges that it is suitable for transplantation when the said GFAP content is 0.5ng or more per 1 mL of said liquid components. The method of claim 1,
Said judgment is an evaluation method of regenerated cartilage which judges that it is suitable for transplantation, when the said GFAP content is 5ng or more per 1 mL of said liquid components. Leaving a cell group including the ear chondrocytes in the presence of a medium,
Thereafter, extracting at least a portion of the liquid component from the medium,
Measuring the content of GFAP contained in the collected liquid component,
Determining whether regenerated cartilage obtained from the cell population is suitable for transplantation based on the GFAP content, and
Forming regenerated cartilage
Method for producing regenerated cartilage comprising a. The method of claim 5, wherein
Forming the regenerated cartilage, the method of producing regenerated cartilage comprising the three-dimensional culture of chondrocytes using a scaffold material. Regenerated cartilage manufactured by the manufacturing method of Claim 5 or 6.

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